This application exploits the recent observation that lymphocytes are readily modified in an enzymatic reaction catalyzed by sortase. Any entity bearing a suitably exposed LPXTG motif can be ligated to the cell surface by means of endogenous acceptor proteins, which must display (an) exposed glycine residue(s) to participate in the reaction. We propose to redirect the specificity of cytotoxic T cells through installation of specific recognition modules that can ultimately be used to selectively target cancerous cells. If successful, this approach would enable the modification ex vivo of cytotoxic T cells or natural killer cells with antibodies or their derivatives that recognize tumor-specific antigens. Reinfusion of such modified autologous cells might then be used as immunotherapy, with several advantages: no genetic modification of the transferred cells is required, the transferred cells are autologous, the enzymatic modification is both rapid and mild, and the elicited response is self-limiting. To pursue this exciting new approach we propose the following specific aims: Aim 1. Functionalization of the lymphocyte cell surface by sortase A-mediated conjugation. Our results show that sortase A can be used to conjugate (modified) peptides or proteins to molecules expressed at the surface of lymphocytes. We shall determine reaction conditions that maximize the number of modified molecules (e.g. sortase variants, cell and probe concentrations, kinetics) as well as identity the endogenous substrates. Aim 2. In vitro cytotoxicity conferred by installation of VHHs on CD8 T cells of known specificity. We have already achieved the installation of single chain antibodies to the murine lymphocyte cell surface using sortase A. We shall investigate whether CD8 T cell cytotoxicity can be redirected by conjugating a single domain antibody fragment of known specificity (anti flu HA, anti Class II MHC). Antigen-specific killing will be assessed in in vitro cytotoxicity assays. Aim 3. In vivo function of redirected lymphocytes. Based on the results from aim 2, we will investigate the potential of this approach to induce killing of tumor cells in a murine tumor model in vivo. If successful, these studies will provide a new form of immunotherapy that exploits the advantages of the specificity of immune recognition, while avoiding genetic modification of the autologous cells to be used as cytotoxic agents. This would be a major advance in cellular immunotherapy of cancer.